The long-term goal of this program is to use murine transplantation models to address mechanistic questions about requirements for, and barriers to, tolerance. Despite great advances, very few strategies induce reproducible tolerance in stringent mouse models and in primates. Therefore, in this competing renewal for a third funding period, our goals are to define mechanisms of resistance to tolerance, and to develop strategies to overcome them for translation in stringent model, in primates and ultimately humans. Why is tolerance in stringent systems (e.g., skin, large animals) so hard to achieve? Studies from our lab and others using peripheral (i.e., non-bone marrow/thymic) approaches to induce tolerance, have shown that: (1) It is relatively easy to induce long-term graft survival/tolerance in mice harboring only naive T cells; (2) Both deletion and the induction of regulatory cells play key roles in this process; and (3) Memory cells, existing due to specific immunization, heterologous immunity, or as a result of homeostatic proliferation following non-specific T cell depletion, are a potent barrier to tolerance. Based on these findings, our overall theoretical framework is that primary barriers to tolerance are resistance to death by effector/memory T cells, and defects in the homeostasis and/or function of regulatory T cells. We believe we now have the necessary tools and models in hand to formulate and test specific hypothesis predicted by this framework. Aim #1 will determine why homeostatic proliferation is a barrier to tolerance. We will test the hypotheses that this barrier is the result of the differential susceptibility of naive T cells, memory T cells, and regulatory T cells to undergo deletion by anti-T cell reagents, and their subsequent ability to "recover" via homeostatic proliferation, under conditions of lymphopenia. Aim #2 will test the susceptibility of memory CD4 T cells to death and regulation. Using a TCR transgenic MHC class II alloreactive CD4 T cell system, we will examine two separate hypotheses, namely that memory T cells are more resistant to death (hypothesis 1) and to regulation (hypothesis 2) than their naive counterparts. Aim #3 will determine whether defects in immunoregulation are mechanisms of tolerance resistance. Using alloreactive CD4+ TCR transgenic mice we will test the hypothesis that failure to acquire or maintain tolerance is due to lack of regulation, relating to either the antigen specificity of the regulatory cell generated, the availability, or lack thereof, of indirect allorecognition, the inherent immunogenicity of selected tissues/organs, and the degree of antigenic disparity between donor and recipient. These studies to define mechanisms of resistance to tolerance in defined clinically relevant models should help in developing novel approaches to induce tolerance in humans.